Revisiting astrocyte to neuron conversion with lineage tracing in vivo.

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.

A single factor elicits multilineage reprogramming of astrocytes in the adult mouse striatum.

SignificanceOutside the neurogenic niches, the adult brain lacks multipotent progenitor cells. In this study, we performed a series of in vivo screens and reveal that a single factor can induce resident brain astrocytes to become induced neural progenitor cells (iNPCs), which then generate neurons, astrocytes, and oligodendrocytes. Such a conclusion is supported by single-cell RNA sequencing and multiple lineage-tracing experiments. Our discovery of iNPCs is fundamentally important for regenerative medicine since neural injuries or degeneration often lead to loss/dysfunction of all three neural lineages. Our findings also provide insights into cell plasticity in the adult mammalian brain, which has largely lost the regenerative capacity.

LncRNA HOTTIP facilitates osteogenic differentiation in bone marrow mesenchymal stem cells and induces angiogenesis via interacting with TAF15 to stabilize DLX2.

AIM: The molecular mechanism of differentiation in bone marrow mesenchymal stem cells (BMSCs) preserves to be further elucidated. LncRNA HOTTIP has been proven to accelerate osteogenic differentiation, but the regulation mechanism is still unclear. METHODS: The human BMSCs (hBMSCs) were isolated and identified by the antigen CD29, CD34, CD44, CD45, and CD90 through flow cytometry. The osteogenic state was determined by the ALP Detection Kit and Alizarin red staining. The tube formation was observed under a microscope. HOTTIP expression level, DLX2 and TAF15, Wnt/ß-catenin pathway, and transcriptional markers in osteogenesis and angiogenesis were examined with Western blot and RT-qPCR, respectively. The combination of TAF15 with lncRNA HOTTIP and DLX2 was detected by RNA immunoprecipitation (RIP) and RNA pulldown assays. RESULTS: The outcomes revealed that HOTTIP was noticeably up-regulated accompanied by the osteogenic transcriptional factor in the process of osteoblast differentiation and angiogenesis. Besides, HOTTIP enhanced alkaline phosphatase (ALP) activity, accelerated osteogenic differentiation and angiogenesis along with up-regulation of osteogenic and angiogenic-related gene expression, by interaction with TAF15 to stabilize DLX2. CONCLUSION: Taken together, our outcomes reveal that lncRNA HOTTIP accelerated osteogenic differentiation and angiogenesis by interaction with TAF15 to stabilize DLX2.

JMJD3 is Involved in Intracranial Aneurysm Development by Regulating DLX2 Expression through H3K27me3 Modification.

Intracranial aneurysms are dilatations in the arteries that supply blood to the brain. Rupture of an intracranial aneurysm leads to a subarachnoid hemorrhage, which is fatal in about 50% of the cases. Microarray-based mRNA expression studies provide unbiased information about molecular mechanisms of intracranial aneurysm and the foundation for functional studies. In this study, by using a Gene Expression Omnibus (GEO) dataset, we identified distal-less homeobox 2 (DLX2) as a significantly upregulated gene in intracranial aneurysms and set to dissect its functional role and upstream mechanism. Here, we found that DLX2 expression was elevated in intracranial aneurysm patients. Silencing of DLX2 suppressed the proliferative capacity of human aortic vascular smooth muscle cells (HA-VSMC) and promoted their apoptosis. Moreover, loss of DLX2 promoted collagen I and collagen III and inhibited the levels of MMP2/9 and pro-inflammatory factors. Additionally, jumonji domain-containing protein 3 demethylase (JMJD3) promoted DLX2 expression by inhibiting H3K27me3 modification. Depletion of JMJD3 exerted the same function as DLX2 in vitro and in vivo, whereas overexpression of DLX2 in the presence of JMJD3 knockdown led to accentuated intracranial aneurysm progression and enhanced HA-VSMC survival. We conclude that JMJD3 promotes DLX2 expression through inhibition of H3K27me3 modification, thereby promoting intracranial aneurysm formation.

Repeated Irradiation with γ-Ray Induces Cancer Stemness through TGF-ß-DLX2 Signaling in the A549 Human Lung Cancer Cell Line.

Cancer stem cells (CSCs) play an important role in cancer recurrence and metastasis. It is suggested that the CSC properties in heterogeneous cancer cells can be induced by ionizing radiation (IR). This study investigated the role of DLX2 in the radioresistance and CSC properties induced by IR in NSCLC cancer cells. Here, A549 cells were exposed to fractionated irradiation at a cumulative dose of 52 Gy (4 Gy × 13 times) for a generation of radioresistant cells. After fractionated irradiation, surviving A549 cells exhibited resistance to IR and enhanced expression of various cancer stem cell markers. They also showed upregulation of mesenchymal molecular markers and downregulation of epithelial molecular markers, correlating with an increase in the migration and invasion. Fractionated irradiation triggered the secretion of TGF-ß1 and DLX2 expression. Interestingly, the increased DLX2 following fractionated irradiation seemed to induce the expression of the gene for the EGFR-ligand betacellulin via Smad2/3 signaling. To contrast, DLX2 knockdown dramatically decreased the expression of CSC markers, migration, and proliferation. Moreover, A549 cells expressing DLX2 shRNA formed tumors with a significantly smaller volume compared to those expressing control shDNA in a mouse xenograft assay. These results suggest that DLX2 overexpression in surviving NSCLC cancer cells after fractionated IR exposure is involved in the cancer stemness, radioresistance, EMT, tumor survival, and tumorigenic capability.

Knockdown of circ_HIPK3 inhibits tumorigenesis of hepatocellular carcinoma via the miR-582-3p/DLX2 axis.

Hepatocellular carcinoma (HCC) is the most common type in the sub-classification of liver cancer. Circular RNAs (circRNAs) play a fundamental role in tumor occurrence and progression. This research aimed to investigate the role and molecular basis of circRNA homeodomain-interacting protein kinase 3 (circ_HIPK3) in HCC. Circ_HIPK3 and DLX2 levels were enhanced, and miR-582-3p level was reduced in HCC tissues and cells. Silencing of circ_HIPK3 impeded proliferation, migration and invasion and expedited apoptosis in HCC cells. Furthermore, circ_HIPK3 modulated HCC progression via sponging miR-582-3p, and miR-582-3p suppressed HCC progression via targeting DLX2. Moreover, circ_HIPK3 knockdown inhibited tumor growth in vivo. Circ_HIPK3 facilitated HCC progression by mediating miR-582-3p/DLX2 pathway, suggesting a new potential biomarker for HCC treatment.

Long non-coding RNA TUG1 promotes cell progression in hepatocellular carcinoma via regulating miR-216b-5p/DLX2 axis.

BACKGROUND: Accumulating evidence indicates that the long noncoding RNA taurine upregulated gene 1(TUG1) plays a critical role in cancer progression and metastasis. However, the overall biological role and clinical significance of TUG1 in hepatocellular carcinoma (HCC) remain largely unknown. METHODS: The expressions of TUG1, microRNA-216b-5p and distal-less homeobox 2 (DLX2) were detected by Quantitative real-time polymerase chain reaction (qRT-PCR). The target relationships were predicted by StarBase v.2.0 or TargetScan and confirmed by dual-luciferase reporter assay. The cell growth, apoptosis, migration and invasion were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Flow cytometry and Transwell assays, respectively. All protein expression levels were detected by western blot. Tumor xenografts were implemented to explore the role of TUG1 in vivo. RESULTS: We found that there was a marked rise in TUG1 expression in HCC tissues and cells, and knockdown of TUG1 repressed the growth and metastasis and promoted apoptosis of HCC cells. In particular, TUG1 could act as a ceRNA, effectively becoming a sink for miR-216b-5p to fortify the expression of DLX2. Additionally, repression of TUG1 impared the progression of HCC cells by inhibiting DLX2 expression via sponging miR-216b-5p in vitro. More importantly, TUG1 knockdown inhibited HCC tumor growth in vivo through upregulating miR-216b-5p via inactivation of the DLX2. CONCLUSION: TUG1 interacting with miR-216b-5p contributed to proliferation, metastasis, tumorigenesis and retarded apoptosis by activation of DLX2 in HCC.

Dlx1/2 are Central and Essential Components in the Transcriptional Code for Generating Olfactory Bulb Interneurons.

Generation of olfactory bulb (OB) interneurons requires neural stem/progenitor cell specification, proliferation, differentiation, and young interneuron migration and maturation. Here, we show that the homeobox transcription factors Dlx1/2 are central and essential components in the transcriptional code for generating OB interneurons. In Dlx1/2 constitutive null mutants, the differentiation of GSX2+ and ASCL1+ neural stem/progenitor cells in the dorsal lateral ganglionic eminence is blocked, resulting in a failure of OB interneuron generation. In Dlx1/2 conditional mutants (hGFAP-Cre; Dlx1/2F/- mice), GSX2+ and ASCL1+ neural stem/progenitor cells in the postnatal subventricular zone also fail to differentiate into OB interneurons. In contrast, overexpression of Dlx1&2 in embryonic mouse cortex led to ectopic production of OB-like interneurons that expressed Gad1, Sp8, Sp9, Arx, Pbx3, Etv1, Tshz1, and Prokr2. Pax6 mutants generate cortical ectopia with OB-like interneurons, but do not do so in compound Pax6; Dlx1/2 mutants. We propose that DLX1/2 promote OB interneuron development mainly through activating the expression of Sp8/9, which further promote Tshz1 and Prokr2 expression. Based on this study, in combination with earlier ones, we propose a transcriptional network for the process of OB interneuron development.

Dlx2 overexpression enhanced accumulation of type II collagen and aggrecan by inhibiting MMP13 expression in mice chondrocytes.

Genetic studies revealed a crucial role of Distal-homebox (Dlx) genes in skeletal development, and our previous study demonstrated overexpressing Dlx2 in neural crest cells led to abnormal cartilage structure, including ectopic cartilage in the maxillary region and nasal bone in mice. The aim of this study was to investigate how Dlx2 overexpression affects chondrogenesis in mouse chondroblast cell line TMC23 and the underlying mechanism. We first demonstrated that Dlx2 expression was upregulated during chondrogenesis in TMC23 cells. Moreover, forced overexpression of Dlx2 in TMC23 cells led to increased accumulation of aggrecan and type II collagen, markers of early chondrocyte differentiation, but had little effect on mRNA and protein levels of Aggrecan and Col2α1, type II collagen gene. Importantly, Dlx2 overexpression decreased mRNA and protein levels of MMP13, a major collagenase degrading aggrecan and type II collagen during late stages of chondrogenesis. Luciferase-reporter and Chromatin-immunoprecipitation analysis demonstrated that MMP13 promoter contained two Dlx2-response elements, and Dlx2 inhibited MMP13 expression by directly binding to these two elements. Based on these observations, we propose that forced overexpression of Dlx2 enhances early chondrocyte differentiation by increasing accumulation of type II collagen and aggrecan, but interferes later stages of chondrocyte differentiation through inhibiting MMP13 expression.

Extended Production of Cortical Interneurons into the Third Trimester of Human Gestation.

In humans, the developmental origins of interneurons in the third trimester of pregnancy and the timing of completion of interneuron neurogenesis have remained unknown. Here, we show that the total and cycling Nkx2.1(+)and Dlx2(+)interneuron progenitors as well as Sox2(+)precursor cells were higher in density in the medial ganglionic eminence (MGE) compared with the lateral ganglionic eminence and cortical ventricular/subventricular zone (VZ/SVZ) of 16-35 gw subjects. The proliferation of these progenitors reduced as a function of gestational age, almost terminating by 35 gw. Proliferating Dlx2(+)cells were higher in density in the caudal ganglionic eminence (CGE) compared with the MGE, and persisted beyond 35 gw. Consistent with these findings, Sox2, Nkx2.1, Dlx2, and Mash1 protein levels were higher in the ganglionic eminences relative to the cortical VZ/SVZ. The density of gamma-aminobutyric acid-positive (GABA(+)) interneurons was higher in the cortical VZ/SVZ relative to MGE, but Nkx2.1 or Dlx2-expressing GABA(+)cells were more dense in the MGE compared with the cortical VZ/SVZ. The data suggest that the MGE and CGE are the primary source of cortical interneurons. Moreover, their generation continues nearly to the end of pregnancy, which may predispose premature infants to neurobehavioral disorders.

Early Craniofacial Defects in Zebrafish That Have Reduced Function of a Wnt-Interacting Extracellular Matrix Protein, Tinagl1.

OBJECTIVE: Tinagl1 has a weak genetic association with craniosynostosis, but its functions in cartilage and bone development are unknown. Knockdown of Tinagl1 in zebrafish embryos allowed an initial characterization of its potential effects on craniofacial cartilage development and a test of whether these effects could involve Wnt signaling. RESULTS: Tinagl1 knockdown resulted in dose-dependent reductions and defects in ventral pharyngeal arch cartilages as well as the ethmoid plate, a zebrafish correlate to the palate. These defects could be correlated to reduced numbers of cranial neural crest cells in the pharyngeal arches and could be reproduced with comanipulation of Tinagl1 and Wnt3a by morpholino-based knockdown. CONCLUSIONS: These results suggest that Tinagl1 is required early in the proliferation or migration of cranial neural crest cells and that its effects are mediated via Wnt3a signaling. Because Wnt3a is among the Wnts that contribute to nonsyndromic cleft lip and cleft palate in mouse and man, further investigation of Tinagl1 may help to elucidate mechanisms underlying these disorders.

Osteocyte-derived exosomes regulate the DLX2/wnt pathway to alleviate osteoarthritis by mediating cartilage repair.

BACKGROUND: Chondrocyte viability, apoptosis, and migration are closely related to cartilage injury in osteoarthritis (OA) joints. Exosomes are identified as potential therapeutic agents for OA. OBJECTIVE: This study aimed to investigate the role of exosomes derived from osteocytes in OA, particularly focusing on their effects on cartilage repair and molecular mechanisms. METHODS: An injury cell model was established by treating chondrocytes with IL-1ß. Cartilage repair was evaluated using cell counting kit-8, flow cytometry, scratch test, and Western Blot. Molecular mechanisms were analyzed using quantitative real-time PCR, bioinformatic analysis, and Western Blot. An OA mouse model was established to explore the role of exosomal DLX2 in vivo. RESULTS: Osteocyte-released exosomes promoted cell viability and migration, and inhibited apoptosis and extracellular matrix (ECM) deposition. Moreover, exosomes upregulated DLX2 expression, and knockdown of DLX2 activated the Wnt pathway. Additionally, exosomes attenuated OA in mice by transmitting DLX2. CONCLUSION: Osteocyte-derived exosomal DLX2 alleviated IL-1ß-induced cartilage repair and inactivated the Wnt pathway, thereby alleviating OA progression. The findings suggested that osteocyte-derived exosomes may hold promise as a treatment for OA.

Efficient Dlx2-mediated astrocyte-to-neuron conversion and inhibition of neuroinflammation by NeuroD1.

In vivo astrocyte-to-neuron (AtN) conversion induced by overexpression of neural transcriptional factors has great potential for neural regeneration and repair. Here, we demonstrate that a single neural transcriptional factor, Dlx2, converts mouse striatal astrocytes into neurons in a dose-dependent manner. Lineage-tracing studies in Aldh1l1-CreERT2 mice confirm that Dlx2 can convert striatal astrocytes into DARPP32+ and Ctip2+ medium spiny neurons (MSNs). Time-course studies reveal a gradual conversion from astrocytes to neurons in 1 month, with a distinct intermediate state in between astrocytes and neurons. Interestingly, when Dlx2-infected astrocytes start to lose astrocytic markers, the other local astrocytes proliferate to maintain astrocytic levels in the converted areas. Unexpectedly, although Dlx2 efficiently reprograms astrocytes into neurons in the gray matter striatum, it also induces partial reprogramming of astrocytes in the white matter corpus callosum. Such partial reprogramming of white matter astrocytes is associated with neuroinflammation, which can be suppressed by the addition of NeuroD1. Our results highlight the importance of investigating AtN conversion in both the gray matter and white matter to thoroughly evaluate therapeutic potentials. This study also unveils the critical role of anti-inflammation by NeuroD1 during AtN conversion.

Effects of Dlx2 overexpression on the genes associated with the maxillary process in the early mouse embryo.

The transcription factor Dlx2 plays an important role in craniomaxillofacial development. Overexpression or null mutations of Dlx2 can lead to craniomaxillofacial malformation in mice. However, the transcriptional regulatory effects of Dlx2 during craniomaxillofacial development remain to be elucidated. Using a mouse model that stably overexpresses Dlx2 in neural crest cells, we comprehensively characterized the effects of Dlx2 overexpression on the early development of maxillary processes in mice by conducting bulk RNA-Seq, scRNA-Seq and CUT&Tag analyses. Bulk RNA-Seq results showed that the overexpression of Dlx2 resulted in substantial transcriptome changes in E10.5 maxillary prominences, with genes involved in RNA metabolism and neuronal development most significantly affected. The scRNA-Seq analysis suggests that overexpression of Dlx2 did not change the differentiation trajectory of mesenchymal cells during this development process. Rather, it restricted cell proliferation and caused precocious differentiation, which may contribute to the defects in craniomaxillofacial development. Moreover, the CUT&Tag analysis using DLX2 antibody revealed enrichment of MNT and Runx2 motifs at the putative DLX2 binding sites, suggesting they may play critical roles in mediating the transcriptional regulatory effects of Dlx2. Together, these results provide important insights for understanding the transcriptional regulatory network of Dlx2 during craniofacial development.

GABAergic neurons differentiated from BDNF- and Dlx2-modified neural stem cells restore disrupted neural circuits in brainstem stroke.

BACKGROUND: Brainstem stroke causes severe and persistent neurological impairment. Due to the limited spontaneous recovery and regeneration of the disrupted neural circuits, transplantation of exogenous neural stem cells (NSCs) was an alternative, while there were limitations for primitive NSCs. METHODS: We established a mouse model of brainstem stroke by injecting endothelin in the right pons. Brain-derived neurotrophic factor (BDNF)- and distal-less homeobox 2 (Dlx2)-modified NSCs were transplanted to treat brainstem stroke. Transsynaptic viral tracking, immunostaining, magnetic resonance imaging, behavioral testing, and whole-cell patch clamp recordings were applied to probe the pathophysiology and therapeutic prospects of BDNF- and Dlx2-modified NSCs. RESULTS: GABAergic neurons were predominantly lost after the brainstem stroke. No endogenous NSCs were generated in situ or migrated from the neurogenesis niches within the brainstem infarct region. Co-overexpressions of BDNF and Dlx2 not only promoted the survival of NSCs, but also boosted the differentiation of NSCs into GABAergic neurons. Results from transsynaptic virus tracking, immunostaining, and evidence from whole-cell patch clamping revealed the morphological and functional integration of the grafted BDNF- and Dlx2-modified NSCs-derived neurons with the host neural circuits. Neurological function was improved by transplantation of BDNF- and Dlx2-modified NSCs in brainstem stroke. CONCLUSIONS: These findings demonstrated that BDNF- and Dlx2-modified NSCs differentiated into GABAergic neurons, integrated into and reconstituted the host neural networks, and alleviated the ischemic injury. It thus provided a potential therapeutic strategy for brainstem stroke.

A Neural Crest-specific Overexpression Mouse Model Reveals the Transcriptional Regulatory Effects of Dlx2 During Maxillary Process Development.

Craniofacial morphogenesis is a complex process that requires precise regulation of cell proliferation, migration, and differentiation. Perturbations of this process cause a series of craniofacial deformities. Dlx2 is a critical transcription factor that regulates the development of the first branchial arch. However, the transcriptional regulatory functions of Dlx2 during craniofacial development have been poorly understood due to the lack of animal models in which the Dlx2 level can be precisely modulated. In this study, we constructed a Rosa26 site-directed Dlx2 gene knock-in mouse model Rosa26 CAG-LSL-Dlx2-3xFlag for conditionally overexpressing Dlx2. By breeding with wnt1 cre mice, we obtained wnt1 cre ; Rosa26 Dlx2/- mice, in which Dlx2 is overexpressed in neural crest lineage at approximately three times the endogenous level. The wnt1 cre ; Rosa26 Dlx2/- mice exhibited consistent phenotypes that include cleft palate across generations and individual animals. Using this model, we demonstrated that Dlx2 caused cleft palate by affecting maxillary growth and uplift in the early-stage development of maxillary prominences. By performing bulk RNA-sequencing, we demonstrated that Dlx2 overexpression induced significant changes in many genes associated with critical developmental pathways. In summary, our novel mouse model provides a reliable and consistent system for investigating Dlx2 functions during development and for elucidating the gene regulatory networks underlying craniofacial development.

Long noncoding RNA distal-less homeobox 2 antisense 1 restrains inflammatory response and apoptosis of periodontal ligament cells by binding with microRNA-330-3p to regulate Ro60, Y RNA binding protein.

OBJECTIVE: This study aims to investigate the role of long noncoding RNA distal-less homeobox 2 antisense 1 (DLX2-AS1) in lipopolysaccharide-induced inflammatory response and apoptosis of periodontal ligament cells (PDLCs). DESIGN: Lipopolysaccharide was used to induce inflammation response of PDLCs. The expression of DLX2-AS1, microRNA-330-3p and Ro60, Y RNA binding protein (RO60) in lipopolysaccharide-treated PDLCs was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Enzyme linked immunosorbent assay (ELISA) was performed to evaluate the concentration of inflammatory cytokines in PDLCs after DLX2-AS1 overexpression or RO60 downregulation. The apoptosis of PDLCs after lipopolysaccharide treatment or indicated transfection was analyzed by flow cytometry analysis. The level of apoptosis-related proteins, Bax and Bcl-2, were examined by western blotting. The binding capacity between microRNA-330-3p and DLX2-AS1 (or RO60) was verified by luciferase reporter assays. RESULTS: DLX2-AS1 was downregulated in PDLCs after lipopolysaccharide treatment. DLX2-AS1 overexpression decreased the production of inflammatory cytokines and inhibited cell apoptosis. microRNA-330-3p bound with DLX2-AS1 and displayed high expression in lipopolysaccharide-induced PDLCs. In addition, the downregulation of RO60, a target gene of microRNA-330-3p, reversed the suppressive influence of DLX2-AS1 overexpression on the inflammatory response and apoptosis of PDLCs. CONCLUSIONS: DLX2-AS1 restrains inflammatory response and apoptosis of PDLCs via the microRNA-330-3p/RO60 axis.

In vitro differentiation of human bone marrow stromal cells into neural precursor cells using small molecules.

BACKGROUND: Neurogenic differentiation of human marrow stromal stem cells (hMSCs) into neural precursor cells (NPCs) offers new hope in many neurological diseases. Stromal cells can be differentiated into NPCs using small molecules acting as chemical inducers. The aim of this study is to formulate an efficient, direct, fast and safe protocol to differentiate hMSCs into NPCs using different inducers: b-mercaptoethanol (BME), triiodothyronine (T3), and curcumin (CUR). NEW METHOD: hMSCs were subjected to either 1 mM BME, 0.5 µM T3, or 5 µM CUR. Neurogenic differentiation was determined by assessing the protein expression of PAX6, SOX2, DLX2, and GAP-43 with flow cytometry and immunofluorescence, along with Nissl staining of differentiated cells. RESULTS AND COMPARISON WITH EXISTING METHOD: It was revealed that T3 and CUR are 70-80% better than BME in terms of efficiency and safety, and surprisingly BME was a good promoting factor for cell preconditioning with limited effects on neural trans-differentiation related to its toxic effects on cell viability. CONCLUSION: Reprogramming of bone marrow stromal cells into neural cells gives hope for treating different neurological disorders. Our study shows that T3 and CUR were effective in generation of NPCs from hMSCs with preservation of cell viability. BME was a good promoting factor for cell preconditioning with limited effects on neural transdifferentiation related to its toxic effects on cell viability.

Partial duplication of the jaw: case reports and review of relevant publications.

Craniofacial duplication is a rare congenital malformation with a wide phenotypic range. The signs and symptoms range from partial craniofacial duplication to bicephalus. We describe two cases of partial duplication of jaw: a girl with a duplication of the maxilla, and a boy with duplication of the mandible. We review the relevant publications and discuss the pathogenesis.

DLX2 activates Wnt1 transcription and mediates Wnt/ß-catenin signal to promote osteogenic differentiation of hBMSCs.

OBJECTIVES: The molocular mechanism underlying human bone marrow mesenchymal stem cells (hBMSCs) differentiation remains to be further elucidated. DLX2 has been confirmed to accelerate osteogenic differentiation which is one member of Distal-less family genes. However, how DLX2 regulates in osteogenic differentiation is still unclear. METHODS: The hBMSCs were isolated and identified by the antigen CD29, CD4, CD90 through flow cytometry. DLX2 expression level, molecules related signaling pathways and transcriptional markers in osteogenesis were examined by western blot and real time-PCR. Osteogenic state was weighed by the ALP Detection Kit and Alizarin red S staining. The combination between DLX2 and WNT1 was detected by Chromatin immunoprecipitation (CHIP) assay. RESULTS: The results showed that in the process of osteoblast differentiation, DLX2 was up-regulated accompanied with osteogenic transcriptional factor. DLX2 elevated cellular alkaline phosphatase activity, accelerated BMSC mineralization along with up-regulation of osteogenic-related gene expression. Besides, DLX2 is a transcription factor of WNT1, which activated the Wnt/ß-Catenin signaling pathway resulting in osteogenic differentiation. Whereas, the inhibitor of ß-Catenin FH535 restrained enhanced osteogenic capability induced by DLX2. CONCLUSIONS: Taken together, these results suggest that by up-regulation of Wnt/ß-Catenin signaling, DLX2 accelerated human osteogenic differentiation.